Methods for the treatment of inflammatory joint disease

ABSTRACT

This invention provides compositions and methods for preventing inflammatory diseases of the joints, including rheumatoid and osteoarthritis, tendonitis, bursitis, inflammation of the ligament, synovitis, gout, and systemic lupus erythematosus, wherein the methods include injecting into the inflamed joint a therapeutic anti-inflammatory composition comprising a bacterial or viral IL-10 expression construct, wherein the IL-10 expression construct comprises a bacterial or viral backbone and a nucleic acid sequence encoding interleukin-10.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority under 35U.S.C. § 120 to U.S. patent applications, U.S. Ser. No. 14/905,915,filed Jan. 18, 2016, which is a national stage filing under 35 U.S.C. §371 of international PCT application, PCT/US2014/047071, filed Jul. 17,2014, which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/847,851, filed Jul. 18, 2013, each of which is incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant numberU44-NS071642 awarded by the National Institute of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to treating clinical conditions associated withinflammatory diseases of the joints and symptoms associated therewith byadministering to the inflamed joint a therapeutic anti-inflammatorycomposition comprising a bacterial or viral interleukin-10 (IL-10)expression construct.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underapplicable statutory provisions.

Joint pain can flare up for any number of reasons, as a result of, e.g.,over a hundred different arthritic conditions—of which rheumatoidarthritis and osteoarthritis are the most common—as well as tendonitis,bursitis, inflammation of the ligament, synovitis, gout, and systemiclupus erythematosus. When injured, a chain of events in the immunesystem known as the inflammatory cascade is triggered, causing redness,swelling and pain. Next, anti-inflammatory compounds take over to healthe area once the threat is diminished. When this process—known as localor acute inflammation takes place—it is a sign of a healthy immunesystem. However, if inflammation persists it can lead to a more chroniccondition.

Current treatments for inflammatory diseases of the joint such asrheumatoid and osteo-arthritis, tendonitis, synovitis and the likeremain suboptimal. Identifying treatments that would require lessfrequent administration would impact significantly the quality of lifefor patients with inflammatory joint disease; however, despitesubstantial research into and development of therapies for suchconditions there is still a large unmet need for safe, effective, andeasy-to-administer treatments.

SUMMARY OF THE INVENTION

The present invention provides methods for treating inflammatorydiseases of the joints, symptoms associated with inflammatory diseasesof the joints, and slowing disease progression by administering to asubject, typically injected into the joint, a vector expressing aninterleukin-10 (IL-10) coding sequence. In some embodiments, the IL-10expression construct is encapsulated in biodegradable microparticles,and in certain aspects of these embodiments, the microparticles aresuspended in a diluent to form a therapeutic composition.

In yet other, alternative embodiments, the therapeutic compositions ofthe present invention are IL-10 expression constructs—with either abacterial or viral backbone—that are delivered as “naked” DNA. That is,the IL-10 expression constructs are delivered without encapsulation. Insuch embodiments, the IL-10 expression constructs are delivered inconjunction with one or more diluents, and in preferred aspects of theseembodiments, the IL-10 expression construct is delivered in conjunctionwith one or more adjuvants. In some aspects of this embodiment, the oneor more adjuvants may be administered to a subject at the time of theadministration of the IL-10 expression construct or up to 10 days beforeadministration of the IL-10 expression construct, e.g., as a“pretreatment”.

In some aspects, the joint inflammation is in the knee, elbow, wrist,ankle, hip, shoulder, or spine. Conditions treatable by the methods ofthe present invention include rheumatoid arthritis and osteoarthritis,as well as tendonitis, bursitis, inflammation of the ligament,synovitis, gout, and systemic lupus erythematosus.

Thus, some embodiments of the invention provide methods for treatment ofinflammatory diseases of the joints or disorders or symptoms associatedtherewith in a subject comprising administering to the subject atherapeutic anti-inflammatory IL-10 compound comprising: plasmid DNAcomprising a bacterial backbone and at least one IL-10 coding sequenceand a diluent; where the therapeutic anti-inflammatory IL-10 compoundprovides a therapeutically effective dose to a joint at about 1 μg DNAto about 1000 μg DNA, or about 5 μg DNA to about 900 μg DNA, or about 10μg DNA to about 850 μg DNA, or about 20 μg DNA to about 800 μg DNA, orabout 25 μg DNA to about 750 μg DNA, or about 40 μg DNA to about 500 μgDNA, or about 50 μg DNA to about 250 μg DNA, or about 5 μg DNA to about200 μg DNA, though preferably about 2.5 μg DNA to about 500 μg DNA. Inother embodiments, a viral vector is used as an alternative to abacterial vector.

In some preferred embodiments of the present invention, the IL-10therapeutic compounds of the present invention (i.e., encapsulated ornaked DNA) are administered with one or more adjuvants. The adjuvants ofthe invention are biocompatible agents that may be administeredsimultaneously with the IL-10 therapeutic compounds or as apre-treatment before the IL-10 therapeutic compounds are administered.Adjuvants that are particularly preferred include but are not limited tomannose, sucrose, glucose, calcium phosphate, dendrimers, liposomesincluding cationic liposomes, and oligodeoxynucleotides. The adjuvantsof the present invention in preferred embodiments are those adjuvantsthat increase uptake or efficacy of the IL-10 expression construct.Concurrent administration or pretreatment includes administering to thejoint about 5 μg adjuvant to about 1000 μg adjuvant, or about 10 μgadjuvant to about 750 μg adjuvant, or about 50 μg adjuvant to about 500μg adjuvant, or about 25 μg adjuvant to about 750 μg adjuvant at thetime of or up to 10 days prior to the administration of the therapeuticanti-inflammatory IL-10 compound.

Other alternative embodiments provide methods for treatment ofinflammatory diseases of the joints comprising administering to thesubject a therapeutic anti-inflammatory composition comprising: an IL-10expression construct; microparticles encapsulating the IL-10 expressionconstruct; and a diluent; where the therapeutic microparticlecomposition provides a therapeutically effective dose to a joint atabout 20 μg DNA to about 1000 μg DNA, or about 25 μg DNA to about 750 μgDNA, or about 50 μg DNA to about 500 μg DNA, or about 50 μg DNA to about250 μg DNA, or about 50 μg DNA to about 200 μg DNA, or or about 25 μgDNA to about 100 μg DNA.

Yet other embodiments of the present invention provide methods forslowing progression of inflammatory diseases of the joints in a subjectcomprising administering to the subject a therapeutic anti-inflammatoryIL-10 compound comprising: plasmid DNA comprising a bacterial backboneand at least one IL-10 coding sequence, where the therapeutic IL-10expression construct provides a therapeutically effective dose to ajoint at about 1 μg DNA to about 1000 μg DNA, about 5 μg DNA to about900 μg DNA, about 10 μg DNA to about 850 μg DNA, about 20 μg DNA toabout 800 μg DNA, or about 25 μg DNA to about 750 μg DNA, or about 50 μgDNA to about 500 μg DNA, or about 50 μg DNA to about 250 μg DNA, orabout 50 μg DNA to about 200 μg DNA, or about 25 μg DNA to about 100 μgDNA. In such embodiments, an adjuvant preferably is administered withthe therapeutic anti-inflammatory IL-10 compound or up to 10 days beforeadministration of the therapeutic anti-inflammatory IL-10 compound,e.g., as a pre-treatment.

Optionally in the embodiments described, the IL-10 expression constructcomprises at least one nuclear targeting sequence located either from100 to 2000 bp 5′ to the at least one IL-10 coding sequence and/or from150 to 450 bp 3′ to the at least one IL-10 coding sequence. In otheraspects of the present invention, the IL-10 expression constructcomprises two nuclear targeting sequences where one nuclear targetingsequence is positioned from 100 to 2000 bp 5′ of the at least one IL-10coding sequence, and one nuclear targeting sequence is positioned from150 to 450 bp 3′ of the at least one coding sequence.

Preferably, the therapeutic composition is administered by injectioninto the joint(s), and in preferred embodiments the therapeuticcomposition is administered by intra-articular injection.

In some aspects, the nucleic acid sequence encoding interleukin-10 hasan amino acid substitution for wildtype phenylalanine at amino acidposition 129, and in some aspects, the amino acid substitution isselected from the group of serine, alanine, threonine or cysteine. Insome aspects, the nucleic acid sequence encoding interleukin-10 encodesIL-10^(F129S). In yet other aspects, the IL-10 expression constructcomprises at least one nuclear targeting sequence 5′ to the IL-10 codingsequence, and in some aspects, IL-10 expression construct comprises atleast one nuclear targeting sequence 3′ to the IL-10 coding sequence.

In some aspects of the present invention, the microparticles compriseone or more of poly(2-hydroxy ethyl methacrylate), poly(N-vinylpyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol),poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate),poly(ethylene glycol), poly(methacrylic acid), polylactides (PLA),polyglycolides (PGA), poly(lactide-co-glycolides) (PLGA),polyanhydrides, polycaprolactone, poly-3-hydroxybutyrate orpolyorthoesters. In certain aspects of the present invention, themicroparticles comprise PLGA. In other aspects of the invention, themicroparticles comprise a mixture of biodegradable polymers representingdifferent, complimentary release profiles.

In certain embodiments of the methods, the therapeutic anti-inflammatoryIL-10 expression construct (and any adjuvant or “pretreatment”adjuvant), is delivered approximately every 40 to 120 days as needed fortherapeutic effect, e.g., up to one year. In other embodiments, thetherapeutic anti-inflammatory composition is delivered approximatelyevery 40 to 120 days as needed for therapeutic effect for greater thanone year. In yet other embodiments, the therapeutic anti-inflammatorycomposition is delivered as needed for therapeutic effect approximatelyevery 40 to 120 days, as needed, for the life of the subject.

The methods of the present invention also may be employed as a researchtool to identify pharmaceuticals, small molecules and/or biologics thatmay be used in conjunction in a “cocktail” with the therapeutic IL-10expression construct compositions.

DESCRIPTION OF THE FIGURES

FIG. 1 shows results in the form of bar graphs illustrating clinicalassessment of the functional improvement and pain reduction achievedtreating osteoarthritis of forelimb joints in canines afteradministration of the therapeutic IL-10 expression constructs of thepresent invention.

FIG. 2 shows results in the form of bar graphs illustrating ownerassessment of the functional improvement and pain reduction achievedtreating osteoarthritis of forelimb joints in canines afteradministration of the therapeutic IL-10 expression constructs of thepresent invention.

FIG. 3 shows results in the form of bar graphs illustrating clinicalassessment of the functional improvement and pain reduction achievedtreating osteoarthritis of forelimb joints in canines afteradministration of the therapeutic IL-10 expression constructs of thepresent invention (pooled data).

FIG. 4 shows results in the form of bar graphs illustrating ownerassessment of the functional improvement and pain reduction achievedtreating osteoarthritis of forelimb joints in canines afteradministration of the therapeutic IL-10 expression constructs of thepresent invention (pooled data).

FIG. 5 shows results illustrating improvements in range of motionachieved treating osteoarthritis of forelimb joints in canines afteradministration of the therapeutic IL-10 expression constructs of thepresent invention (pooled data).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, publishedpatent applications and patents mentioned herein are incorporated byreference in their entirety for the purpose of describing and disclosingdevices, animal models, formulations and methodologies that may be usedin connection with the presently described invention.

The term adjuvant as used herein refers to a pharmacological orimmunological agent that modifies the effect of other agents. In thecontext of the present invention, an adjuvant is used to increase theefficacy of the therapeutic IL-10 anti-inflammatory compound. Adjuvantsof particular utility in the present invention include those thatenhance the uptake or efficacy of the IL-10 expression construct by,e.g., macrophages or other immune cells present in the synovial fluid ofthe joint.

The term “anti-inflammatory” as used herein refers to decreasing theaction or production of one or more proinflammatory cytokines orproteins produced by nerves, neurons, glial cells, endothelial cells,fibroblasts, muscle, immune cells or other cell types.

The term “anti-inflammatory cytokine” as used herein refers to a proteinthat decreases the action or production of one or more proinflammatorycytokines or proteins produced by nerves, neurons, glial cells,endothelial cells, fibroblasts, muscle, immune cells or other cell typesInflammatory cytokines and proteins include, without limitation,interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-a),interleukin-6 (IL-6), inducible nitric oxide synthetase (iNOS) and thelike. Non-limiting examples of anti-inflammatory cytokines includeinterleukin-10 (IL-10) including viral IL-10, interleukin-4 (IL-4),interleukin-13 (IL-13), alpha-MSH, transforming growth factor-beta 1(TGFβ1), and the like. Thus, the full-length molecules and fragments ofanti-inflammatory cytokines, as well as anti-inflammatory cytokines withmodifications, such as deletions, additions and substitutions (eitherconservative or non-conservative in nature), to the native sequence, areintended for use herein, so long as the anti-inflammatory cytokine istherapeutically effective. Modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the proteins or errors due to PCRamplification. Accordingly, active proteins are typically substantiallyhomologous to the parent sequence, e.g., proteins are typically about 70. . . 80 . . . 85 . . . 90 . . . 95 . . . 98 . . . 99%, etc. homologousto the parent sequence.

A “coding sequence” of an anti-inflammatory cytokine or a sequence that“encodes” an anti-inflammatory cytokine is a nucleic acid molecule thatis transcribed (in the case of DNA) and translated (in the case of mRNA)into a polypeptide in vivo when placed under the control of appropriatecontrol sequences. The boundaries of the coding sequence are determinedby nucleotides corresponding to a start codon at the amino terminus andnucleotides corresponding to a translation stop codon at the carboxyterminus.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, enhancers, and the like, which collectively provide for thereplication, transcription and translation of a coding sequence in arecipient cell. Not all of these types of control sequences need to bepresent so long as the selected coding sequence is capable of beingreplicated, transcribed and translated in an appropriate host cell.

The terms “effective amount” or “therapeutically effective amount” of atherapeutic IL-10 expression construct used in the methods of theinvention refer to a nontoxic but sufficient amount of the IL-10expression construct to provide the desired response, such as a decreasein inflammation of the joints, relief from symptoms caused byinflammatory diseases of the joints and/or preventing progression ofjoint damage due to inflammatory diseases of the joints. The exactamount required will vary from subject to subject, depending on thespecies, age, and general condition of the subject, the severity of thecondition being treated, and the particular IL-10 expression constructto be delivered whether the IL-10 expression construct is delivered in amicroparticle or as naked DNA, mode of administration (e.g.,intra-articular injection), and the like. Dosage parameters for thepresent methods are provided herein; however, optimization of anappropriate “effective” amount in any individual case may be determinedby one of ordinary skill in the art using the methods set forth hereinand routine experimentation.

The term “excipient” or “diluent” refers to an inert substance added toa therapeutic composition of the invention to facilitate administrationof the therapeutic IL-10 expression construct. Examples, withoutlimitation, of excipients include saline, calcium carbonate, calciumphosphate, various sugars and types of starch, cellulose derivatives,gelatin, hyaluronic acid optionally formulated with a surfactant,Plurnoic F-68, vegetable oils and polyethylene glycols.

By “isolated” when referring to a nucleotide sequence, is meant that theexpression construct is present in the substantial absence of otherbiological macromolecules of the same type. Thus, an “isolated nucleicacid molecule which encodes a particular polypeptide” refers to anucleic acid molecule which is substantially free of other nucleic acidmolecules that do not encode the subject polypeptide; however, themolecule may include some additional bases or moieties that do notdeleteriously affect the basic characteristics of the composition.

The term “joint” refers to an anatomical structure where two bones meet,including the ligaments that connect the bones to one another, thetendons that attach muscles to the bones, the joint capsule, bursae andsynovium. Joints that can be treated with the methods herein includefixed, hinge, pivot or ball-and-socket joints.

The term “joint inflammation” refers to all types of arthritis caused byinflammation where rheumatoid arthritis, osteoarthritis are the mostcommon, as well as tendonitis, bursitis, inflammation of the ligament,synovitis, gout, and systemic lupus erythematosus.

The term “nuclear targeting sequence” refers to a nucleic acid sequencewhich functions to improve the expression efficiency of ananti-inflammatory cytokine in a cell.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

The term “promoter” is used herein in its ordinary sense to refer to anucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene that is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence. Transcription promoters can include“inducible promoters” (where expression of a polynucleotide sequenceoperably linked to the promoter is induced by an analyte, cofactor,regulatory protein, etc.), “repressible promoters” (where expression ofa polynucleotide sequence operably linked to the promoter is induced byan analyte, cofactor, regulatory protein, etc.), and “constitutivepromoters”.

For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “3 prime (3′)” or “5 prime(5′)” relative to another sequence, it is to be understood that it isthe position of the sequences in the “sense” or “coding” strand of a DNAmolecule that is being referred to as is conventional in the art.

The term “research tool” as used herein refers to any methods of theinvention using the therapeutic IL-10 expression constructs forscientific inquiry, either academic or commercial in nature, includingthe development of other pharmaceutical and/or biological therapeutics.The research tools of the invention are not intended to be therapeuticor to be subject to regulatory approval; rather, the research tools ofthe invention are intended to facilitate research and aid in suchdevelopment activities, including any activities performed with theintention to produce information to support a regulatory submission.

The terms “subject”, “individual” or “patient” may be usedinterchangeably herein and refer to a vertebrate, preferably a mammal.

The term “therapeutic composition” or “therapeutic anti-inflammatorycomposition” as used herein refers to a composition that has the abilityto decrease inflammation of the joints, provide relief from symptomscaused by inflammatory diseases of the joints and/or prevent progressionof joint damage due to inflammatory diseases of the joints as measuredin any of the known animal models or by assessment performed in humans.

“Treatment” or “treating” joint inflammation includes: (1) decreasinginflammation of the joint or causing the inflammation to occur with lessintensity in a subject that may be predisposed to joint inflammation butdoes not yet experience or display symptoms, or (2) inhibiting jointinflammation, i.e., arresting the development of or reversing symptomsor physiological damage caused by inflammation.

A “viral vector” as used herein is a recombinantly produced virus orviral particle that comprises an IL-10 expression construct to bedelivered into a host cell, either in vivo, ex vivo or in vitro.Examples of viral vectors include retroviral vectors, lentiviralvectors, adenovirus vectors, adeno-associated virus vectors, alphavirusvectors and the like.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and sequencing technology,which are within the skill of those who practice in the art. Specificillustrations of suitable techniques can be had by reference to theexamples herein. However, other equivalent conventional procedures can,of course, also be used. Such conventional techniques and descriptionscan be found in standard laboratory manuals such as LeDoux (Ed.) (2005),Animal Models of Movement Disorders (Academic Press); Chow, et al.,(2008), Using Animal Models in Biomedical Research (World ScientificPublishing Co.); Weir and Blackwell (Eds.), Handbook of ExperimentalImmunology, Vols. I-IV (Blackwell Scientific Publications); Creighton(1993), Proteins: Structures and Molecular Properties (W.H. Freeman andCompany); Sambrook and Russell (2006), Condensed Protocols fromMolecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002),Molecular Cloning: A Laboratory Manual (both from Cold Spring HarborLaboratory Press); Stryer, L. (1995) Biochemistry, Fourth Ed. (W. H.Freeman); Gait (1984), “Oligonucleotide Synthesis: A Practical Approach”(IRL Press); Nelson and Cox (2000), Lehninger, Principles ofBiochemistry, Third Ed. (W. H. Freeman); and Berg et al. (2002)Biochemistry, Fifth Ed. (W. H. Freeman); all of which are hereinincorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

Methods of the Invention

The present invention provides methods for treating inflammatorydiseases of the joints, symptoms associated with inflammatory diseasesof the joints, and slowing disease progression by administering to asubject—typically injected via intra-articular injection—a vectorexpressing a therapeutic interleukin-10 (IL-10) expression construct. Insome embodiments, the IL-10 expression construct is encapsulated inbiodegradable microparticles, and in most aspects of this embodiment,the microparticles are suspended in a diluent to form a therapeuticcomposition. As an alternative, in preferred embodiments the therapeuticIL-10 expression vector is delivered as a “naked” vector, where thedelivery of the naked IL-10 expression vector is accompanied by orpreceded by administration of a nucleic acid uptake adjuvant. Thenucleic acid uptake adjuvant may be administered either concurrently orup to ten or more days prior to the administration of the naked IL-10expression vector. The methods of the present invention may be used totreat joint inflammation in the knee, elbow, wrist, ankle, hip,shoulder, or spine. Conditions treatable by the methods of the presentinvention include rheumatoid arthritis and osteoarthritis, as well astendonitis, bursitis, inflammation of the ligament, synovitis, gout, andsystemic lupus erythematosus.

Thus, the invention generally provides methods for treating inflammatorydiseases of the joints and symptoms and physiological damage associatedwith inflammatory diseases of the joints. The invention also providesfor using the methods of the invention in research of inflammatorydiseases of the joints, including identifying pharmaceuticals, smallmolecules and/or biologics that may be used in conjunction (in a“cocktail”) with the therapeutic IL-10 expression construct. The methodscomprise the step of administering to a subject, preferably by injectioninto a joint, an IL-10 expression construct comprising a bacterial orviral backbone and at least one IL-10 coding sequence. In someembodiments, the IL-10 expression construct is optionally encapsulatedin biodegradable microparticles. The anti-inflammatory compositions aregenerally suspended in a diluent for delivery to a joint. The IL-10expression construct may comprise at least one nuclear targetingsequence where the at least one nuclear targeting sequence is 5′, 3′ orboth to the IL-10 coding sequence.

The anti-inflammatory compounds of the present invention may consist ofa “naked” bacterial vector or viral vector capable of expressing the atleast one IL-10 coding sequence, or the anti-inflammatory compound mayconsist of a bacterial or viral vector encased in microparticles orother delivery device. Administration of the therapeutic IL-10expression construct may be accompanied by or preceded by administrationof an adjuvant, and in the case of delivery of naked IL-10 expressionconstructs, a nucleic acid uptake adjuvant may be administeredconcurrently or up to ten days or more before administration of thenaked IL-10 expression construct.

The IL-10 expression construct used in some embodiments of the methodsof the present invention comprises a bacterial backbone (plasmid DNA orpDNA) or a viral backbone, at least one IL-10 coding sequence, at leastone nuclear targeting sequence 5′ (upstream), 3′ (downstream) or both ofthe at least one IL-10 coding sequence, and one or more DNA controlsequences. Optionally, the pDNA also may comprise one or more additionalanti-inflammatory cytokine coding sequences, and/or a marker sequence toallow for selection of transformed cells during amplification of thepDNA. The bacterial backbone can be any bacterial backbone known tothose with skill in the art. Backbones typically selected are thosethat, e.g., contain or lack appropriate restriction sites to allow easeof cloning, may be produced and isolated with ease, are not immunogenic,and the like. For example, bacterial backbones derived from E. coli areof use in the present invention.

The plasmid DNA comprises at least one IL-10 coding sequence. IL-10coding sequence may code for wildtype IL-10, or the IL-10 may be amutant IL-10. One mutant IL-10 of interest contains one or moremutations that cause amino acid substitutions, additions or deletions ascompared to wildtype IL-10 in the “hinge” region of the IL-10 protein.The human IL-10 protein is a homodimer, where each monomer comprises sixalpha helices A→F, the length of which are 21, 8, 19, 20, 12 and 23amino acids, respectively. Helices A→D of one monomer noncovalentlyinteract with helices E and F of a second monomer, forming a noncovalentV-shaped homodimer. The “hinge” region targeted for mutation accordingto the present invention comprises the amino acids between the D and Ealpha helices on one or both monomers at approximately amino acidposition X to position Y of wildtype IL-10. For example, mutant rat andhuman IL-10 proteins have been described in which the phenylalanine atposition 129 of the wildtype sequence has been replaced with a serineresidue. (See, e.g., Sommer, et al., WO2006/130580 and Milligan, et al.,Pain, 126:294-308 (2006).) The resulting mutant IL-10 is referred to asIL-10^(F129S). Other substitutions for the wildtype phenylalanine atamino acid position 129 may be, e.g., threonine, alanine, or cysteine.Thus the present invention in yet another aspect encompasses one or moresubstitutions at amino acid position 129 or at other amino acids withinthe hinge region of the IL-10 protein, or functional equivalentsthereof.

Additional anti-inflammatory cytokines of use in the present inventioninclude but are not limited to interleukin-4 (IL-4), interleukin-13(IL-13), alpha-MSH, transforming growth factor-beta 1 (TGFβ1), and thelike.

Nuclear targeting sequences of the present invention are sequences thatpromote expression of the protein(s) encoded by the at least one IL-10coding sequence and the optional, additional anti-inflammatory cytokinecoding sequence(s). For example, in one aspect the nuclear targetingsequences may bind to nuclear transport chaperone proteins, facilitatinguptake of the plasmid DNA by the cell nucleus. Such sequences includebut are not limited to interspersed (or dispersed) DNA repeats orrepetitive sequences such as transposable elements, flanking or terminalrepeats such as the long terminal repeats (LTRs) on retrovirus genomessuch as SV40s, tandem repeats, and the inverted terminal repeats (ITRs)of viral genomes such as Adeno-Associated Virus and Adenovirus. In otheraspects, the nuclear targeting sequences are sequences that act to bindtranscription factors for import into the nucleus, such as enhancersequences.

In addition to a bacterial backbone, at least one IL-10 coding sequenceand optional one or more additional anti-inflammatory cytokine codingsequences and, optionally, one or more nuclear targeting sequences, theplasmid DNA of the present invention comprises one or more DNA controlsequences, such as promoter sequences, polyadenylation signals,transcription termination sequences, upstream regulatory domains,origins of replication, internal ribosome entry sites and the like,which collectively provide for the replication, transcription andtranslation of the anti-inflammatory cytokine coding sequence(s) in arecipient cell. Not all of these control sequences need always bepresent so long as the anti-inflammatory cytokine coding sequences arecapable of being replicated, transcribed and translated in anappropriate host cell. Promoter sequences of use in the presentinvention include but are not limited to chicken or human (3-actinpromoters, cytomegalovirus immediate early promoters, glyceraldehydes3-phosphate dehydrogenase (GADPH) promoters, elongation factor 1α(cF1α)promoter, GFAP promoter, murine leukemia virus (MLV) promoter, herpessimples virus thymidine kinase (TK) promoter, and woodchuck hepatitisvirus post-transcriptional regulatory element (WPRE) promoters; upstreamregulatory domains of use in the present invention include but are notlimited to cytomegalovirus immediate early promoter enhancers, mousemammary tumor virus (MMTV) enhancer and simian virus 40 (SV40) enhancer;and polyadenylation signals of interest in the present invention includebut are not limited to SV40 polyadenylation signal, bovine growthhormone polyadenylation signal, and synthetic polyadenylation signals.Optionally, the plasmid DNA of the present invention will also comprisea selection marker gene, such as that coding for antibiotic resistance.Marker genes of use in the present invention include but are not limitedto neomycin, hygromycin-B, ampicillin, kanomycin, or puromycin.

For example, plasmids comprising the rat IL-10 sequence flanked by twoAAV ITRs, a cytomegalovirus immediate early promoter enhancer, a chickenβ-actin promoter, a polyadenylation signal, and a herpes simplexthymidine kinase promoter driving a neomycin resistance marker were usedin some experiments demonstrating the usefulness of the presentinvention. In other experiments, plasmids comprising the human IL-10sequence flanked by two AAV ITRs, a cytomegalovirus immediate earlypromoter enhancer, a cytomegalovirus immediate early promoter, apolyadenylation signal, and an ampicillin resistance marker were used.Details of these plasmids are disclosed in Milligan, et al., Pain126:294-308 (2006).

Alternatively, the vector may be a viral vector. In general, the fivemost commonly used classes of viral systems used in gene therapy can becategorized into two groups according to whether their genomes integrateinto host cellular chromatin (oncoretroviruses and lentiviruses) orpersist in the cell nucleus predominantly as extrachromosomal episomes(adeno-associated virus, adenoviruses, herpesviruses, andintegration-deficient lentiviruses). For example, in one embodiment ofthe present invention, viruses from the Parvoviridae family areutilized. The Parvoviridae is a family of small single-stranded,non-enveloped DNA viruses with genomes approximately 5000 nucleotideslong. Included among the family members is adeno-associated virus (AAV),a dependent parvovirus that by definition requires co-infection withanother virus (typically an adenovirus or herpesvirus) to initiate andsustain a productive infectious cycle. In the absence of such a helpervirus, AAV is still competent to infect or transduce a target cell byreceptor-mediated binding and internalization, penetrating the nucleusin both non-dividing and dividing cells.

Another viral delivery system useful with the IL-10 expressionconstructs of the present invention is a system based on viruses fromthe family Retroviridae. Retroviruses comprise single-stranded RNAanimal viruses that are characterized by two unique features. First, thegenome of a retrovirus is diploid, consisting of two copies of the RNA.Second, this RNA is transcribed by the virion-associated enzyme reversetranscriptase into double-stranded DNA. This double-stranded DNA orprovirus can then integrate into the host genome and-be passed fromparent cell to progeny cells as a stably-integrated component of thehost genome.

In some embodiments, lentiviruses are the preferred members of theretrovirus family for use in the present invention. Lentivirus vectorsare often pseudotyped with vesicular stomatitis virus glycoprotein(VSV-G), and have been derived from the human immunodeficiency virus(HIV), the etiologic agent of the human acquired immunodeficiencysyndrome (AIDS); visan-maedi, which causes encephalitis (visna) orpneumonia in sheep; equine infectious anemia virus (EIAV), which causesautoimmune hemolytic anemia and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immunodeficiency virus (BIV) which causes lymphadenopathy andlymphocytosis in cattle; and simian immunodeficiency virus (SIV), whichcauses immune deficiency and encephalopathy in non-human primates.Vectors that are based on HIV generally retain <5% of the parentalgenome, and <25% of the genome is incorporated into packagingconstructs, which minimizes the possibility of the generation ofreverting replication-competent HIV. Biosafety has been furtherincreased by the development of self-inactivating vectors that containdeletions of the regulatory elements in the downstreamlong-terminal-repeat sequence, eliminating transcription of thepackaging signal that is required for vector mobilization. The mainadvantage to the use of lentiviral vectors is that gene transfer ispersistent in most tissues or cell types due to integration of the viralvector. However, lentivirus that is integrase deficient could also beused.

Adenoviruses (Ads) are a relatively well characterized homogenous groupof viruses, including over 50 serotypes. See, e.g., International PCTApplication No. WO 95/27071. Adenoviruses are medium-sized (90-100 nm),nonenveloped (without an outer lipid bilayer) icosahedral virusescomposed of a nucleocapsid and a double-stranded linear DNA genome.There are 57 described serotypes in humans, which are responsible for5-10% of upper respiratory infections in children, and many infectionsin adults as well. They are classified as group I under the Baltimoreclassification scheme, meaning their genomes consist of double-strandedDNA, and are the largest nonenveloped viruses. Because of their largesize, they are able to be transported through the endosome (i.e.,envelope fusion is not necessary). The virion also has a unique “spike”or fiber associated with each penton base of the capsid that aids inattachment to the host cell via the coxsackie-adenovirus receptor on thesurface of the host cell.

The adenovirus genome is linear, non-segmented double-stranded (ds) DNAthat is between 26 and 45 kb, allowing the virus to theoretically carry22 to 40 genes. Although this is significantly larger than other virusesin its Baltimore group, it is still a very simple virus and is heavilyreliant on the host cell for survival and replication. Once the virushas successfully gained entry into the host cell, the endosomeacidifies, which alters virus topology by causing capsid components todisassociate. With the help of cellular microtubules, the virus istransported to the nuclear pore complex, where the adenovirus particledisassembles. Viral DNA is subsequently released, which can enter thenucleus via the nuclear pore. After this the DNA associates with histonemolecules. Thus, viral gene expression can occur and new virus particlescan be generated.

Unlike most lentiviral vectors (i.e., those that can integrate, e.g.,are not integrase deficient), adenoviral DNA does not integrate into thegenome and is not replicated during cell division. The primaryapplications for adenovirus are in gene therapy and vaccination.Recombinant adenovirus-derived vectors, particularly those that reducethe potential for recombination and generation of wild-type virus, havealso been constructed. See, International PCT Application Nos. WO95/00655 and WO 95/11984.

Other viral or non-viral systems known to those skilled in the art alsomay be used to deliver IL-10 expression constructs of the presentinvention to the joint, including but not limited to gene-deletedadenovirus-transposon vectors that stably maintain virus-encodedtransgenes in vivo through integration into host cells (see Yant, etal., Nature Biotech. 20:999-1004 (2002)); systems derived from Sindbisvirus or Semliki forest virus (see Perri, et al., J. Virol.74(20):9802-07 (2002)); systems derived from Newcastle disease virus orSendai virus; or mini-circle DNA vectors devoid of bacterial DNAsequences (see Chen, et al., Molecular Therapy. 8(3):495-500 (2003)).Mini-circle DNA as described in U.S. Patent Publication No. 2004/0214329discloses vectors that provide for persistently high levels of nucleicacid transcription.

Once the IL-10 expression construct has been constructed, amplified andisolated by techniques known in the art, the IL-10 expression constructis then optionally, in some embodiments, encapsulated withinmicroparticles. Techniques for encapsulating IL-10 expression constructvary depending on the type of microparticles used and such techniquesare described in more detail infra. The microparticles of the presentinvention may be comprised of any biodegradable polymer. To be usedsuccessfully as a biodegradable polymer in the controlled drug deliveryformulations of the present invention, the material must be chemicallyinert and free of leachable impurities. Ideally the polymer also has anappropriate physical structure, with minimal undesired aging, and isreadily processable. Some of the materials include poly(2-hydroxy ethylmethacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate),poly(vinyl alcohol), poly(acrylic acid), polyacrylamide,poly(ethylene-co-vinyl acetate), poly(ethylene glycol), andpoly(methacrylic acid). Biodegradable polymers of particular use in thepresent invention include polylactides (PLA), polyglycolides (PGA),poly(lactide-co-glycolides) (PLGA), polyanhydrides, polycaprolactone,poly-3-hydroxybutyrate and polyorthoesters. Such biodegradable polymershave been characterized extensively and can be formulated to exhibitdesired degradation properties as is known in the art (see, e.g., Edlund& Albertsson, Degradable Aliphatic Polyesters, pp. 67-112 (2002),Barman, et al., J. of Controlled Release, 69:337-344 (2000); Cohen, etal., Pharmaceutical Res., (8): 713-720 (1991)).

In one particular embodiment of the invention, the polymer comprisespoly(lactide-co-glycolides) (PLGA). PLGA is a copolymer which is used ina host of FDA approved therapeutic devices, owing to itsbiodegradability and biocompatibility. PLGA is synthesized by means ofrandom ring-opening co-polymerization of two different monomers, thecyclic dimers (1,4-dioxane-2,5-diones) of glycolic acid and lactic acid.Common catalysts used in the preparation of this polymer include tin(II)2-ethylhexanoate, tin(II) alkoxides, or aluminum isopropoxide. Duringpolymerization, successive monomeric units of glycolic or lactic acidare linked together in PLGA by ester linkages, thus yielding a linear,aliphatic polyester as a product.

Depending on the ratio of lactide to glycolide used for thepolymerization, different forms of PLGA can be obtained: these areusually identified in regard to the monomers' ratio used (e.g., PLGA75:25 identifies a copolymer whose composition is 75% (molar percent)lactic acid and 25% (molar percent) glycolic acid). PLGA degrades byhydrolysis of its ester linkages in the presence of water. It has beenshown that the time required for degradation of PLGA is related to themonomers' ratio used in production: the higher the content of glycolideunits, the lower the time required for degradation. An exception to thisrule is the copolymer with 50:50 monomers' ratio which exhibits thefaster degradation (about two months). In addition, polymers that areend-capped with esters (as opposed to the free carboxylic acid)demonstrate longer degradation half-lives. Of particular use in thepresent invention is PLGA having a composition of between 20% and 80%lactic acid and between 80% and 20% glycolic acid. More preferred foruse in the present invention is PLGA having a composition of between 65%and 35% lactic acid and between 35% and 65% glycolic acid. In one aspectof the present invention, PLGA having a composition of 50% lactic acidand 50% glycolic acid is used.

Additionally, the IL-10 expression constructs (pDNA) may be encapsulatedin batches of microparticles having different release profiles; forexample, 10% of the pDNA to be delivered may be encapsulated inmicroparticles having, e.g., a one day to four week release profile; 30%of the pDNA to be delivered may be encapsulated in microparticleshaving, e.g., a three week to six week release profile; 30% of the pDNAto be delivered may be encapsulated in microparticles having, e.g., asix week to ten week release profile; and 30% of the pDNA to bedelivered may be encapsulated in microparticles having, e.g., an eightweek to twelve week release profile. In such an embodiment, a singletype of biodegradable polymer may be used, but used in formulations withdifferent release profiles; alternatively, different biodegradablepolymers having different release characteristics may be used. In yetanother embodiment, the formulation of the microparticles may be variedso as to change the surface of the microparticles to enhance or retard,as desired, the travel of the therapeutic composition through, e.g., thesynovial fluid of the joint.

Once microparticles are obtained, they are suspended in an acceptablediluent to form a therapeutic composition for administration to asubject. Similarly, if the IL-10 expression constructs of the presentinvention are delivered as naked DNA as opposed to being encapsulated,diluents are also used for administration to the subject. Such diluents(or excipients) include any pharmaceutical agent that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition, and that may be administered without undue toxicity.Pharmaceutically acceptable diluents may comprise sorbitol, alum,dextran, sulfate, large polymeric anions, any of the various TWEENcompounds, and liquids such as water, saline, glycerol or ethanol, oiland water emulsions, or adjuvants such as Freund's adjuvant.Pharmaceutically acceptable salts can be included therein as well, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like may be present in suchvehicles. In one aspect of the invention, different diluents are usedbased on their ability to migrate through the targeted site in joint.For example, for delivery in an adult human, diluents may be preferredthat favor more rapid spread of the therapeutic composition through thejoint; conversely, in children or small animals where the size of thejoint is less of a concern, a diluent may be used that does not dispersethe therapeutic composition quickly. A thorough discussion ofpharmaceutically acceptable diluents/excipients is available inRemington: The Science and Practice of Pharmacy, 21^(st) ed., LippincottWilliams & Wilkins (2005). Preferred diluents include but are notlimited to Physiosol®, artificial synovial fluid, preservative-free 0.9%NaCl, lactated Ringer's injection solution, and Elliotts B® Solution.

Synthesizing and administering the compositions to be used in themethods of the present invention involve a series of steps. First, aplasmid is constructed comprising the various components describedsupra. Then the plasmid DNA (pDNA) is amplified and isolated bytechniques well known in the art. Once the pDNA is isolated, it may becombined with polymers to form pDNA-containing microparticles. Methodsfor microparticle formation vary depending on the polymers used,however, a double emulsion technique is typically employed. First, apolymer is dissolved in an organic solvent. Next, pDNA is suspended inan aqueous solution and is added to the polymer solution. The twosolutions are then mixed to form a first emulsion. The solutions can bemixed by vortexing or shaking or by passage through a particulate mediumproducing turbulence, or the mixture can be sonicated. Most preferableis any method by which the nucleic acid receives the least amount ofdamage in the form of nicking, shearing, or degradation, while stillallowing the formation of an appropriate emulsion. During this process,the polymer forms into microparticles, many of which contain pDNA. Ifdesired, one can isolate a small amount of the nucleic acid at thispoint in order to assess integrity, e.g., by gel electrophoresis.

The first emulsion is then added to an organic solution. The solutioncan be comprised of, for example, methylene chloride, ethyl acetate, oracetone, typically containing polyvinyl alcohol (PVA), and often havingapproximately a 1:100 ratio of the weight of PVA to the volume of thesolution. The first emulsion is generally added to the organic solutionwith stirring in a homogenizer or sonicator. This process forms a secondemulsion which is subsequently added to another organic solution withstirring (e.g., in a homogenizer). In one aspect of this method, thelatter solution is 0.05% w/v PVA. The resultant microparticles arewashed several times with water to remove the organic compounds. In someaspects of the present invention, more than approximately 40% of theresulting microparticles contain pDNA. In yet other aspects, more thanapproximately 50% of the resulting microparticles contain pDNA, in yetother aspects of the present invention, more than 55% of the resultantmicroparticles contain pDNA.

The ability to internalize differently-sized microparticles varies withcell type. In certain embodiments of the invention, macrophages andantigen-presenting cells were targeted. Such cells more efficientlyinternalize microparticles of less than about 5μ (see Shakweh, et al.,Eur J of Pharmaceutics and Biopharmaceutics 61(1-2):1-13 (2005)). Thus,if desired, particles may be passed through sizing screens toselectively remove those larger than the desired size. In one particularaspect of the invention, microparticles of less than 5μ are used in thetherapeutic composition, and in other particular aspects of theinvention, microparticles of less than 3μ are used in the therapeuticcomposition. After washing, the particles can either be used immediatelyor be lyophilized for storage. The size distribution of themicroparticles prepared by the methods described herein can bedetermined with, e.g, a Coulter™ counter or laser diffraction.Alternatively, the average size of the particles can be determined byvisualization under a microscope fitted with a sizing slide or eyepiece.Alternatively, a scanning electron microscope can be used to assess bothsize and microparticle morphology.

Once IL-10 expression construct-containing microparticles are obtained,the microparticles can be suspended immediately in diluent orlyophilized for storage. The combination of the microparticles anddiluent forms the therapeutic microparticle composition that can beadministered by injection into a joint to an animal subject. Therecombinant vectors can be introduced either in vivo or in vitro (alsotermed ex vivo) to treat joint inflammation. If transduced in vitro, thedesired recipient cell or synovial fluid is removed from the subject,treated with pDNA-containing microparticles and reintroduced into thesubject. Alternatively, syngeneic or xenogeneic cells can be transformedfor delivery where such cells typically do not generate an inappropriateimmune response in the subject. If administered in vivo, recombinantvectors or cells transformed with the vectors in vitro are delivereddirectly by injection into the joint.

The IL-10 expression constructs of the present invention are, in analternative embodiment, administered as naked DNA. In such anembodiment, the IL-10 expression constructs are amplified, e.g., usinggood quality manufacturing practices. GMPs are enforced in the UnitedStates by the U.S. Food and Drug Administration (FDA), under Section501(B) of the 1938 Food, Drug, and Cosmetic Act (21 USCS § 351).

Adjuvants appropriate for the present invention include adjuvants thatincrease the uptake of the IL-10 expression constructs of the presentinvention; that is, adjuvants appropriate for the present inventioninclude any biologically-compatible agent that neutralizes or obviatesthe issue of introducing negatively-charged DNA into cells with anegatively-charged membrane. Such adjuvants include sugars such asmannose, glucose and sucrose; calcium phosphate; dendrimers(repetitively branched molecules); liposomes (spherical vesiclescomprising a lipid bilayer) including cationic liposomes; DEAE-dextranincluding DEAE-dextran polyethylenimine; oligodeoxynucleotides; and highmolecular weight hyaluronic acid (>1 MDa), an anionic nonsulfatedglycosaminoglycan.

One adjuvant of particular interest is D-mannose. D-mannose is a simplehexose sugar with a molecular weight of 180.2 and is known to: decreaseinflammatory processes during wound healing (Kossi J, et al., Eur SurgRes, 31(1):74-82 (1999), reduce oxidative bursts required duringinflammation (Rest R F, et al., J Leukoc Biol, 43(2):158-164 (1988)),suppress adjuvant-induced arthritis in a rat model (Willenborg D O, etal., Immunol Cell Biol, 70(Pt 6):369-377 (1996)), inhibit LPS-inducedIL-1β, TNF-α, decrease NF-κB/p65 critical for proinflammatory cytokineexpression, and decrease leukocyte influx following intratrachealinstillation of LPS, which is a model of sepsis-associated acute lunginjury and respiratory distress syndrome (Xu X L, et al., Inflamm Res,57(3):104-110 (2008); Xu X, et al., Eur J Pharmacol, 641(2-3):229-2372010)). The MR is a transmembrane glycoprotein pattern recognitionreceptor involved in host defense of innate immunity by recognizingmannosylated ligands (for example, lysosomal hydrolases) that caninclude a variety of bacteria, yeasts and parasites expressingmannosylated molecules (see, e.g., Engering A J, et al., Adv Exp MedBiol, 417:183-187 (1997); Linehan S A, et al., Adv Exp Med Biol,479:1-14 (2000); Stahl P D, et al., Curr Opin Immunol, 10(1):50-55(1998)).

Because the therapeutic compositions of the invention do notsignificantly induce an immune response or dose tolerance in subjects,they can be administered as needed for therapeutic effect. That is, thetherapeutic anti-inflammatory composition can be delivered approximatelyevery 40 to 120 days (or as required) as needed for therapeutic effectfor shorter-term therapy. However, when longer-term therapy is desired,the therapeutic composition can be delivered approximately every 40 to120 days (or more or less) as needed for therapeutic effect for greaterthan one year; and if necessary, for the life of the subject. Dosagefrequency depends on the dosage, the adjuvant used and the health of thesubject.

Dosage ranges of the therapeutic compositions used in the methods of thepresent invention vary from subject to subject, depending on thespecies, age, and general condition of the subject, the severity of thecondition being treated, joint site, and the particular IL-10 expressionconstruct to be delivered, whether or not the IL-10 expression constructif encapsulated, mode of administration, and the like. Dosage rangesinclude a therapeutically effective dose per joint at about 1-1000 μgvector DNA, about 5-750 μg vector DNA, about 10-600 μg vector DNA,20-500 μg vector DNA, 25-250 μg vector DNA, or 50-100 μg vector DNA.

The IL-10 expression constructs or microparticles containing the IL-10expression constructs used in the methods of the present invention maybe co-administered in a “cocktail” with other therapeutic agents usefulin treating joint inflammation including glucocorticoids; methotrexate;hydroxychlolquine; sulfasalazine; lefunomide; anti-TNF agents such asetanercept, infliximab and adalimumab; abatacept; hyaluronic acid,particularly high molecular weight hyaluronic acid (>1 MDa) such asHyalgan, Orthovisc, or Synvisc at a dose of, e.g., 0.5-2.5% (5 to 25mg/mL) from 1 to 5 mL, so from 5 mg to 125 mg per joint; andnonsteroidal anti-inflammatory drugs (NSAIDs). Additionally, the IL-10expression constructs or microparticles containing the IL-10 expressionconstructs used in the methods of the present invention may beco-administered with cells, such as mesenchymal stem cells or other stemcells, including stem cells bioengineered to express IL-10 expressionconstructs. Generally, any method known in the art can be used tomonitor success of treatment in humans, including both clinical andphenotypic indicators.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting. Inthe Examples, procedures that are constructively reduced to practice aredescribed in the present tense, and procedures that have been carriedout in the laboratory are set forth in the past tense.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific embodiments withoutdeparting from the spirit or scope of the invention as broadlydescribed. The present embodiments are, therefore, to be considered inall respects as illustrative and not restrictive.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees centigrade, and pressure is at or nearatmospheric.

Amplification and Purification of pDNA

The plasmid construct encoding for rat interleukin-10 (pDNA-IL-10F129S)has been previously described in detail in Milligan, et al., Pain126(1-3): 294-308 (2006). In short, the plasmid consists of a 5.9Kilobase (Kb) circular plasmid DNA containing a transcriptional cassetteconsisting of a cytomegalovirus enhancer/chicken beta-actin promoter(CMV enh/CB pro) driving expression of the rat IL-10 gene containing apoint mutation (F129S) and a viral SV40 polyadenylation signal. Thetranscription cassette is flanked by a 149 bp inverted terminal repeatsequence. An identical plasmid lacking the IL-10 gene was used as a pDNAcontrol. Both plasmids were amplified in SURE 2 Supercompetent E. colicells (Agilent Technologies, USA) and isolated using an endotoxin freeGiga plasmid purification kit (Qiagen, Valencia, Calif., USA) accordingto the manufacturer's instructions. Purified, endotoxin-free plasmidswere resuspended in sterile Dulbecco's PBS (DPBS, 1, 0.1 micronpore-filtered, pH 7.2, catalog #14190-144; Gibco, Invitrogen Corp, GrandIsland, N.Y., USA) with 3% sucrose (DPBS-3%). The DPBS-3% vehicle wasprepared using molecular biology grade D (+)-sucrose(b-D-fructofuranosyl-a-D-glucopyranoside; Sigma-Aldrich, St. Louis, Mo.,USA) in DPBS, 0.2 um sterile filtered (pyrogen-free syringe filter unit,catalog #25AS020AS, Life Science Products, Inc., CO, USA) and stored insterile, 15 ml conical tubes at 4° C. until the time of use.

Microparticle Preparation and Characterization

Microparticles were prepared using a modified double emulsion/solventevaporation protocol (A. M. Tinsley-Bown, et al., J. of ControlledRelease 66(2-3): 229-41 (2006)). Briefly, a 50:50 PLGA copolymer (MW75,000, Lactel Absorbable Polymers) was dissolved in ethyl acetate(Sigma). Vehicle alone (phosphate buffered saline (PBS) +3% (w/v)sucrose (Sigma)) or pDNA in vehicle were emulsified in the PLGA solutionfollowed by a second emulsion in a 5% (w/v) polyvinyl alcohol, 28%calcium chloride, 3% sucrose (Sigma) and 7% (v/v) ethyl acetatesolution. After 4 hours of hardening in a wash solution, the resultingmicroparticles were collected, lyophilized and stored at 4° C. Scanningelectron microscopy (SEM) was used to examine microparticle morphology.The diameters of >1000 microparticles present in 10 different imageswere measured with NIH ImageJ software and binned particle diameterswere used to generate a normalized frequency distribution. The zetapotential of the microparticles was measured with a Nicomp 380 ZLS ZetaPotential Analyzer, and the endotoxin levels of the resultantmicroparticles were tested by the LAL assay, using serial dilution as acontrol for inhibition. The microparticles utilized exhibited aspherical and smooth morphology under SEM and a zeta-potential of−28.04±2.12 mV. The microparticles exhibited a heterogeneous sizedistribution with an overall median diameter of 4.67±0.26 μm, which isconsistent with similar methods of microparticle manufacturing and thepDNA encapsulation efficiency for the particles was 55.1%.

Total pDNA encapsulation was assessed by extracting pDNA frommicroparticles via sodium hydroxide dissolution, measuring theabsorbance at 260 nm and comparing obtained values to DNA standards atknown concentrations. Final pDNA loadings were 8.78±0.65 μg pDNA/mgPLGAfor PLGA-pDNA-IL-10 microparticles. Aqueous extraction of pDNA wasconducted by dissolving microparticles in chloroform and allowing thepDNA to migrate into aqueous buffer. The extracted pDNA was subsequentlyconcentrated by precipitation with ethanol and re-suspended in PBS+3%sucrose vehicle. The structural integrity of the aqueous extracted pDNAwas compared against unencapsulated pDNA (which was similarly exposed tothe aqueous extraction process) by loading 2 μg of total pDNA into thewells of a 1.0% agarose gel containing ethidium bromide, running the gelat 75 V for 2 hours, and imaging the gel with UV trans-illumination at305 nm. Biological activity of aqueous extracted pDNA was assessed bylipofectamine-mediated transfection into human embryonic kidney-293cells according to manufacturer protocols (Invitrogen) and IL-10 proteinconcentrations in cell culture supernatants collected 24 hours aftertransfection with aqueous extracted and unencapsulated pDNA wereassessed by ELISA (R&D Systems). In vitro release profiling wasconducted by incubating microparticles in PBS over time in a water bathat 37° C. and pDNA contents in the supernatant were quantified by aPicoGreen assay (Milligan, et al., Neuron Glia Biology 2(4) 293-308(2006)).

Agarose gel electrophoresis of aqueous extracted pDNA frommicroparticles compared to unencapsulated pDNA indicated that asignificant amount of the relaxed and supercoiled pDNA structuralintegrity was preserved after encapsulation, although a slight detectionof linearized pDNA and slight alterations in the migration of multimericpDNA species were observed after encapsulation. By comparing resultantIL-10 protein expression levels in the supernatants of human embryonickidney-293 cells 24 hours after lipofectamine-mediated transfection withdose-matched microparticle extracted or unencapsulated pDNA, it wasdetermined that the microparticle extracted pDNA-IL-10 exhibited a 96.8%biological activity retention for the resultant production of IL-10(data not shown). In vitro pDNA release analysis demonstrated that 30%of the pDNA was released after 3 days and steady release was achievedfor greater than 75 days. This two-phase release profile is a commoncharacteristic of macromolecule release from emulsion based PLGAmicroparticles, where the enhanced phase of initial pDNA release is dueto an increased pDNA content on or near the surface of themicroparticles which is followed by a sustained release and diffusion ofpDNA from the microparticle interior (Yeo, Archive of Endotoxin Res.27(1): 1-12 (2004)). Endotoxin levels from microparticles with andwithout encapsulated pDNA were below the limits of detection for the LALassay up to a microparticle concentration of 10 mg/ml (1 mg ofmicroparticles/well).

D-Mannose

In embodiments where an adjuvant such as D-Mannose is employed,D-mannose (catalog # M6020) can be purchased carrier free fromSigma-Aldrich (St. Louis, Mo.). The D-mannose is combined with 0.1% BSAin sterile saline, and administered via intr-articular injection eitherconcurrently with the IL-10 expression construct or from one to ten dayprior to administration of the IL-10 expression construct. D-mannose istypically delivered at a dosage of 2.5 μg-500 μg per joint in canines.

Administration for Treatment of Joint Inflammation in Canines

The IL-10 expression construct (pDNA-IL-10^(F129S)) as “naked” pDNA wasadministered by acute intra-articular injection to an affected joint ina series of canines. Under IV sedation and anesthetic monitoring, thecanine patient's affected joint was surgically clipped to remove all furover the affected joint and sterilized utilizing a surgical scrub, suchas chlorahexidine 2%. The patient's heart rate, blood pressure, oxygensaturation, ventilation and heart rhythm was continuously monitored. A22- or 20-gauge hypodermic needle was inserted into the synovial space.Synovial fluid was aspirated prior to administration of the pDNA toassure proper placement of the needle. The syringe containing synovialfluid aspirate was replaced while maintaining intra-articular needleplacement. Once the synovial fluid was aspirated, the therapeuticanti-inflammatory composition was administered into the joint utilizingthe same intra-articular needle. Up to 1 mg plasmid DNA equivalent wasinjected, although as little as 700 μg was found to be effective. Wherethe volume per joint injection during administration did exceed 1 ml,joint fluid was aspirated to compensate. Following the successfulplacement of the therapeutic anti-inflammatory composition within thejoint space, the patient was reversed from the sedative affects andclinically monitored. Any changes to blood pressure, heart rate,oxygenation, ventilation or heart rhythm during the procedure wascorrected with the proper medical treatments. Anticipated effects ofsedation include bradycardia, hypotension, hypoventilation. Oxygentherapy was continuous throughout the entire procedure to maximizeoxygen saturation levels. Sedative medications tailoring to individualpatient and sole medications or combinations of the following:

A: Dexmedetomidine 0.5 mg/m², Reversal (atipamazole 0.05-0.2 mg/lb)

B: Opioids—Butorphanol (0.05-0.1 mg/lb),

C: Propofol—i.v. to effect

D: Benzodiazepines (diazepam): 0.1-0.2 mg/lb

-   Subjects remained at the veterinary facility for the day (less than    12 hours) and then were allowed go home.

Clinical assessments included owner Canine Brief Pain Inventorybehavioral assessments, veterinarian clinical Visual Analog Scale forpain and mobility, goniometry, pharmaceutical reduction/dependency, andvideo and gait monitoring. FIG. 1 shows results in the form of bargraphs illustrating clinical assessment of the functional improvementand pain reduction achieved treating osteoarthritis of forelimb jointsin canines after administration of the therapeutic IL-10 expressionconstructs of the present invention. Note that administration of theIL-10 expression constructs of the present invention resulted insignificant functional improvement and pain reduction, in all of walk,trot, manipulation, range of motion and functional disability,particularly at the 11-week mark.

FIG. 2 shows results in the form of bar graphs illustrating ownerassessment of the functional improvement and pain reduction achievedtreating osteoarthritis of forelimb joints in canines afteradministration of the therapeutic IL-10 expression constructs of thepresent invention. Note that again, there was significant improvement inall parameters of activity, quality of life, rising, walking, runningand climbing stairs even at 1 week.

FIG. 3 shows results in the form of bar graphs illustrating clinicalassessment of the functional improvement and pain reduction achievedtreating osteoarthritis of forelimb joints in canines afteradministration of the therapeutic IL-10 expression constructs of thepresent invention (pooled data). The results of clinical assessment showsignificant positive results, particularly in pain.

FIG. 4 shows results in the form of bar graphs illustrating ownerassessment of the functional improvement and pain reduction achievedtreating osteoarthritis of forelimb joints in canines afteradministration of the therapeutic IL-10 expression constructs of thepresent invention (pooled data). Note in these results, similar to theresults in FIG. 2, there was significant improvement in all parametersof activity, quality of life, rising, walking, running and climbingstairs even at 1 week.

FIG. 5 shows results illustrating improvements in range of motionachieved treating osteoarthritis of forelimb joints in canines afteradministration of the therapeutic IL-10 expression constructs of thepresent invention (pooled data). Note that the change in angle degreesshowed significant improvement.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

We claim:
 1. A method for treating inflammatory joint disease in asubject, said method comprising injecting into the inflamed joint atherapeutic anti-inflammatory composition comprising a therapeuticallyeffective amount of a bacterial or viral IL-10 expression construct,wherein the IL-10 expression construct comprises a bacterial or viralbackbone and a nucleic acid sequence encoding interleukin-10.
 2. Themethod of claim 1, wherein the IL-10 expression construct isadministered with an adjuvant.
 3. The method of claim 2, wherein theadjuvant is selected from D-mannose, sucrose, glucose, calciumphosphate, dendrimers, oligonucleotides, high molecular weighthyaluronic acid, or liposomes.
 4. The method of claim 1, wherein thenucleic acid sequence encoding interleukin-10 has an amino acidsubstitution for wildtype phenylalanine at amino acid position
 129. 5.The method of claim 4, wherein the amino acid substitution is selectedfrom the group of serine, alanine, threonine or cysteine.
 6. The methodof claim 5, wherein the nucleic acid sequence encoding interleukin-10encodes IL-10^(F129S).
 7. The method of claim 1, wherein the plasmid DNAcomprises at least one nuclear targeting sequence 5′ to the IL-10 codingsequence.
 8. The method of claim 1, wherein the plasmid DNA comprises atleast one nuclear targeting sequence 3′ to the IL-10 coding sequence. 9.The method of claim 1, further comprising a diluent.
 10. The method ofclaim 1, wherein the joint is a knee, elbow, wrist, ankle, hip,shoulder, or spine.
 11. The method of claim 1, wherein the inflammatoryjoint disease is arthritis, tendonitis, bursitis, inflammation of theligament, synovitis, gout, and systemic lupus erythematosus.
 12. Amethod for treating inflammatory joint disease in a subject, said methodcomprising injecting into the inflamed joint a therapeuticanti-inflammatory composition comprising a therapeutically effectiveamount of a bacterial or viral IL-10 expression construct, wherein theIL-10 expression construct comprises a bacterial or viral backbone and anucleic acid sequence encoding interleukin-10; and microparticlesencapsulating the expression construct.
 13. The method of claim 12,wherein the nucleic acid sequence encoding interleukin-10 has an aminoacid substitution for wildtype phenylalanine at amino acid position 129.14. The method of claim 13, wherein the amino acid substitution isselected from the group of serine, alanine, threonine or cysteine. 15.The method of claim 14, wherein the nucleic acid sequence encodinginterleukin-10 encodes IL-10^(F129S).
 16. The method of claim 10,wherein the polymer comprises poly(lactic-co-glycolic acid).
 17. Themethod of claim 16, wherein the polymer comprises 50:50poly(lactic-co-glycolic acid).
 18. The method of claim 10, wherein theplasmid DNA comprises at least one nuclear targeting sequence 5′ to theIL-10 coding sequence.
 17. The method of claim 10, further comprising adiluent.
 18. The method of claim 10, wherein the joint is a knee, elbow,wrist, ankle, hip, shoulder, or spine.
 19. The method of claim 10,wherein the inflammatory joint disease is arthritis, tendonitis,bursitis, inflammation of the ligament, synovitis, gout, and systemiclupus erythematosus.
 20. The method of claim 19, wherein theinflammatory joint disease is osteoarthritis.
 21. A method for treatinginflammatory joint disease in a subject, said method comprisinginjecting into the inflamed joint a therapeutic anti-inflammatorycomposition comprising 1-500 μg of a bacterial or viral IL-10 expressionconstruct, wherein the IL-10 expression construct comprises a bacterialor viral backbone and a nucleic acid sequence encoding interleukin-10;and 5-1000 μg D-mannose.
 22. The method of claim 21, wherein theD-mannose is administered concurrently with the IL-10 expressionconstruct.
 23. The method of claim 21, wherein the De-mannose isadministered up to ten days prior to administration of the IL-10expression construct.
 24. The method of claim 22, wherein the nucleicacid sequence encoding interleukin-10 has an amino acid substitution forwildtype phenylalanine at amino acid position
 129. 25. The method ofclaim 24, wherein the amino acid substitution is selected from the groupof serine, alanine, threonine or cysteine.
 26. The method of claim 25,wherein the nucleic acid sequence encoding interleukin-10 encodesIL-10^(F129S).
 27. The method of claim 22, wherein the plasmid DNAcomprises at least one nuclear targeting sequence 5′ to the IL-10 codingsequence.
 28. The method of claim 22, wherein the plasmid DNA comprisesat least one nuclear targeting sequence 3′ to the IL-10 coding sequence.29. The method of claim 22, wherein the plasmid DNA comprises at leastone nuclear targeting sequence both 5′ and 3′ to the IL-10 codingsequence.
 30. A method for treating inflammatory joint disease in asubject, said method comprising injecting into the inflamed joint atherapeutic anti-inflammatory composition comprising 1-500 μg of abacterial or viral IL-10 expression construct, wherein the IL-10expression construct comprises a bacterial or viral backbone, a nucleicacid sequence encoding IL-10^(F129S), at least one nuclear targetingsequence either 5′ or 3′ or both to the IL-10 coding sequence; and5-1000 μg D-mannose.